Method for detecting methylated dna

Patent metadata

Field	Value
Publication number	US-2016138097-A1
Application number	US-201414771936-A
Country	US
Kind code	A1
Filing date	Mar 7, 2014
Priority date	Mar 7, 2013
Publication date	May 19, 2016
Grant date	—

Abstract

Provided is a rapid and simple method of detecting methylated DNA. The method of detecting methylated DNA includes the following steps of: (1) treating sample DNA with a hydrogen sulfite; (2) amplifying the sample DNA treated with the hydrogen sulfite by PCR; and (3) subjecting the resultant PCR amplification product to ion-exchange chromatography.

First claim

1 . A method of detecting methylated DNA, comprising (1), (2), and (3):
(1) treating sample DNA with a hydrogen sulfite; (2) amplifying the sample DNA treated with the hydrogen sulfite by PCR; and (3) subjecting the resultant PCR amplification product to ion-exchange chromatography.

2 . A method of detecting methylated DNA, comprising (1), (2), and (3′):
(1) treating sample DNA with a hydrogen sulfite;
(2) amplifying the sample DNA treated with the hydrogen sulfite by PCR; and
(3′) subjecting the resultant PCR amplification product to ion-exchange chromatography at a column temperature of 45° C. or more and less than 90° C.

3 . The method according to claim 1 , wherein the ion-exchange chromatography comprises anion-exchange chromatography.

4 . The method according to claim 1 , wherein a column packing material to be used in the ion-exchange chromatography contains comprises base particles each having both a strong cationic group and a weak cationic group on a surface thereof on its surface.

5 . The method according to claim 1 , wherein an eluent used in elution of the PCR amplification product in the ion-exchange chromatography comprises an antichaotropic ion.

6 . The method according to claim 5 , wherein the antichaotropic ion comprises any one or both of a sulfate ion and an ammonium ion.

7 . The method according to claim 1 , further comprising the step of comparing a detection signal obtained in the ion-exchange chromatography of the PCR amplification product of the sample DNA treated with the hydrogen sulfite to a detection signal obtained in ion-exchange chromatography of:
a PCR amplification product of DNA treated with the hydrogen sulfite, the DNA being identical in nucleotide sequence to the sample DNA but being free of methylation; or a PCR amplification product of DNA treated with the hydrogen sulfite, the DNA being identical in nucleotide sequence to the sample DNA and having a predetermined proportion of methylation.

8 . The method according to claim 7 , further comprising measuring, based on results of the comparing step, presence or absence, a methylation ratio, or a presence ratio of methylated DNA in the sample DNA.

9 . The method according to claim 1 , further comprising subtracting a detection signal obtained in ion-exchange chromatography of a PCR amplification product of DNA treated with the hydrogen sulfite, the DNA being identical in nucleotide sequence to the sample DNA but being free of methylation from a detection signal obtained in the ion-exchange chromatography of the PCR amplification product of the sample DNA treated with the hydrogen sulfite to obtain differential data.

10 . The method according to claim 9 , further comprising measuring, based on the differential data, presence or absence, a methylation ratio, or a presence ratio of methylated DNA in the sample DNA.

11 . A method of extracting a signal of methylated DNA from a signal of sample DNA, the method comprising (1) to (6):
(1) treating sample DNA with a hydrogen sulfite; (2) amplifying the sample DNA treated with the hydrogen sulfite by PCR; (3) subjecting a PCR amplification product obtained in (2) to ion-exchange chromatography; (4) treating DNA that is identical in nucleotide sequence to the sample DNA but is free of methylation with the hydrogen sulfite, followed by PCR amplification; (5) subjecting a PCR amplification product obtained in (4) to ion-exchange chromatography; and (6) subtracting a detection signal in chromatography obtained in (5) from a detection signal in chromatography obtained in (3) to obtain differential data.

12 - 13 . (canceled)

Assignees

• Sekisui Medical Co Ltd
• Univ Mie

Inventors

• Yotani Takuya
• Nobori Tsutomu

Classifications

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• G01N30/96

  using ion-exchange (G01N30/02, G01N30/90 take precedence) · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title